Stroke and vascular dementia are prevalent diseases which are both caused by multiple factors, including genetic susceptibility. In large part, the genetics of these disorders is complex. However, in families with CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), premature stroke and vascular dementia are caused by defects in the Notch3 gene. Our broad goal is to identify the molecular pathways governing the pathogenesis of CADASIL, which will help shed light on more common forms of stroke and dementia. The central hypothesis is that mutant NotchS, expressed in arterial smooth muscle, triggers a series of cellular changes that result in inhibition of the proteasome. The proteasome is essential for the degradation of proteins via the ubiquitin proteasome system and has been implicated in several neurodegenerative disorders. We propose that inhibition of the proteasome in vascular smooth muscle causes cell toxicity and is linked to the breakdown of cell-cell contacts and accumulation of Notch3 protein, two pathological features of CADASIL. In support of this concept, we present preliminary data showing that Notch3 specifically interacts with a subunit of the 26S proteasome. In addition, we demonstrate evidence that proteasome inhibition is increased in cells expressing mutant Notch3. Finally, proteasome inhibition in cultured smooth muscle cells causes cell-cell separation and cell death, two features seen in CADASIL tissues. In Specific Aim 1, we will characterize whether mutant Notch3 directly inhibits proteasomes and whether mutant Notch3 over-expression leads to proteasome inhibition in cell culture and in arteries of transgenic animals. In Aim 2, we will determine whether proteasome inhibition leads to cell-cell contact disruption and Notch3 protein accumulation in culture and in vivo. Finally, in Aim 3, we will investigate whether cell death due to proteasome inhibition is exacerbated by Notch3 mutations and by cell-cell contact disruption. These experiments will support a CADASIL pathway involving proteasomal inhibition and may have implications for the pathogenesis of other vascular and proteasome-linked diseases. In lay language, this application is designed to investigate the molecules involved in a disease, CADASIL that causes stroke and dementia. An understanding of CADASIL may provide clues to designing better treatments and prevention strategies for stroke and dementia. What is learned from this study may also provide information that will be useful for the treatment of other vascular diseases.